The present invention relates to a virus capable of specifically infecting and growing within red tide plankton; a method and an agent for preventing red tide using the virus; and a method for isolating the virus and a method for subculturing the virus. More specifically, the present invention relates to a virus capable of specifically infecting and growing within a member of algae Heterocapsa sp., a method and an agent for preventing red tide using the virus; and a method for isolating the virus and a method for subculturing the virus.
In Japan, the yield from sea surface culture makes up approximately one quarter of the whole yield of the national fishery industry. To promote the sea surface culture, the environmental protection of culture fisheries is indispensable, and above all, it is extremely important to take effective countermeasures against red tide causing severe damage to commercial fisheries.
A red tide-causing alga, Heterocapsa circularisquama, which appeared in Japan in the late 80s, is a kind of dinoflagellate having the property of specifically killing bivalves such as oysters, short-necked clams and pearl oysters, and results in severe financial losses to the bivalve-culture industries of many regions in Japan. In 1998, losses to the culture oyster industry in Hiroshima Bay alone reached approx. 3.8 billion yen. In 1988, when Heterocapsa red tide was recorded for the first time in the world, Heterocapsa cells were observed only in Uranouchi Bay located in Kochi prefecture. However, since then the distribution area of Heterocapsa has rapidly expanded, and at present it is widely distributed throughout the coastal regions of western Japan from Kumamoto prefecture to Fukui prefecture. Fortunately, the movement of this species to neighboring countries has not been reported yet, but the international transport of hazardous and toxic plankton by means of the ballast water of freighters is known, and so the monitoring system of the distribution dynamics of this species needs to be improved.
Against this backdrop, municipalities in each region in Japan strongly desire establishment of concrete measures against Heterocapsa red tide. At this stage, however, there are no concrete measures apart from xe2x80x9cthe displacement of culture farmsxe2x80x9d. However, the displacement of culture farms from red tide emerging areas is replete with potential danger that Heterocapsa cells are also displaced. Therefore, culturists are required to displace their culture farms with extra care.
Aiming at the development of practical techniques for preventing Heterocapsa red tide, the present invention is mainly directed to the search for possible applications of a novel isolated virus, Heterocapsa circularisquama Virus (HcV) to prevent red tide. The prevention of red tide by utilizing the virus is expected to be especially useful, and is highly regarded as an environmental protection technique, which is safe and reduces burdens on other living things or on the whole ecosystem. So, if some challenges, e.g. development of mass culture technique and dispersion method of the virus, and achievement of low cost, are resolved, a technique using this virus is expected to proceed toward commercialization in the future.
After performing thorough studies to overcome the above-stated problems, the present inventors have succeeded in isolating for the first time in the world a virus which infects and kills Heterocaspa circularisquama, thereby completing the present invention.
That is to say, the present invention provides a virus capable of specifically infecting and growing within a member of algae Heterocapsa sp. This Heterocapsa sp. can be Heterocapsa circularisquama. In one aspect of the present invention, the virus lacks a tail and an adventitia, adopts a sphere-like regular icosahedral structure with a particle size of approx. 0.1 to 0.2 xcexcm, and has double-stranded DNA.
In addition, the present invention provides a method for isolating a virus capable of specifically infecting and growing within a member of algae Heterocapsa sp., which comprises a process of: filtrating with a filter a liquid sample containing a member of algae Heterocapsa sp. infected with a virus capable of specifically infecting and growing within the above Heterocapsa sp.; inoculating the obtained filtrate into a culture solution of a member of algae Heterocapsa sp. and culturing; and cloning the virus by performing a limiting dilution on a culture solution wherein the above Heterocapsa sp. is observed to be lysed. The pore size of the filter used may be large enough to trap living things larger than bacteria or viruses such as phytoplankton, and for isolating HcV for example, the pore size of filters used may be 0.4 or 0.2 xcexcm. Regardless of whether a member of algae Heterocapsa sp. is cultured before or after infection with the virus of the present invention, the culture is preferably performed at a temperature of 20 to 30xc2x0 C., photon density of 40 to 70 xcexcmol photons mxe2x88x922 s-1, and under a light-dark cycle. Furthermore, the culture may be performed in modified SWM3 medium. As preferable culture conditions, temperature is 20xc2x0 C., photon density is 45 xcexcmol photons mxe2x88x922 s-1, and the light-dark cycle is a 12-hour cycle. Limiting dilution performed during a process of cloning the virus of the present invention may be carried out in a series of 10xe2x88x921 to 10xe2x88x927 times dilution steps.
Moreover, the present invention provides an agent for preventing red tide, which comprises, as an active ingredient, a virus capable of specifically infecting and growing within a member of algae Heterocapsa sp.
Furthermore, the present invention provides a method for preventing red tide, which comprises dispersing a virus capable of specifically infecting and growing within a member of algae Heterocapsa sp. in red tide-fouled waters.
In addition, the present invention provides a method for subculturing a virus capable of specifically infecting and growing within a member of algae Heterocapsa sp., which comprises a process of repeating at least once the following steps: centrifuging the culture solution of a member of algae Heterocapsa sp., wherein cell lysis is observed as a result of infection with a virus capable of specifically infecting and growing within the above Heterocapsa sp.; inoculating the obtained supernatant into a culture solution of a member of algae Heterocapsa sp.; and culturing. Regardless of whether a member of algae Heterocapsa sp. is cultured before or after infection with the virus of the present invention, the culture thereof is preferably performed at a temperature of 20 to 30xc2x0 C., photon density of 40 to 70 xcexcmol photons mxe2x88x922 s-1, and under a light-dark cycle. Furthermore, the culture may be performed in modified SWM3 medium. As preferable culture conditions, temperature is 20xc2x0 C., photon density is 45 xcexcmol photons mxe2x88x922 s-1, and the light and dark cycle is a 12-hour cycle.
Throughout the present specification, xe2x80x9cred tidexe2x80x9d means the development of discoloration of sea water caused by plankton during drastic population increases. This word is widely used, regardless of whether there is damage to aquatic living things presents or not. Representative examples of plankton causing harmful red tide in Japan include Chattonella sp., Gymnodinium sp., Heterosigma sp., etc.
xe2x80x9cA member of algae Heterocapsa sp.xe2x80x9d belongs to Dinophyceae, and among them, species known to belong to Heterocapsa sp. include Heterocapsa circularisquama, Heterocapsa triquetra, Heterocapsa illdefina, etc.
xe2x80x9cHeterocapsa circularisquamaxe2x80x9d is a relatively small dinoflagellate (approx. 20 xcexcm in dimension) having the property of specifically killing shellfish, while not affecting other Pisces. In 1988, red tide caused by this species occurred for the first time in Uranouchi Bay located in Kochi prefecture, and it resulted in severe losses, killing 1,500 tons of short-necked clams. Since then, red tides caused by this species have expanded throughout western Japan, and severe damage to the fishery industry occurs almost every year. Since the toxicity of this species to shellfish is specific and lethal, there is a possibility that an unknown mechanism of the species causes the death of shellfish. For this reason, even now several thorough studies are being conducted in order to clarify the mechanism of the toxicity of this species.
xe2x80x9cVirusxe2x80x9d means a spherical or fibrous microstructure capable of growing only within a host cell infected with the same. Viruses are completely different from bacteria in that it is incapable of growing by binary fission, but capable of self-replicating only by utilizing the biosynthetic system of a host cell. When compared with bacteria, viruses are characterized by having significantly high host specificity. xe2x80x9cHost specificityxe2x80x9d is an expression indicating the limitation of types of host organisms which pathogenic parasites (which are viruses herein) are able to infect and grow within. For instance, an expression such as to xe2x80x9chave high host specificityxe2x80x9d or to xe2x80x9chave narrow host rangexe2x80x9d means that there are a few kinds of organisms which parasites are able to infect.